CN115040693B - Containing CD56 + Biological material of exosome from subcellular group and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a material containing CD56 ⁺ A biological material of exosomes derived from subcellular groups and a preparation method thereof. Comprising the following steps: methacrylic acid anhydride gelatin, spinal cord acellular matrix and exosomes; the exosomes are derived from a polypeptide bearing CD56 ⁺ A labeled cell subpopulation; the exosome expresses signalin 7A. The exosome has a plurality of protein types for promoting the regeneration of the nerve axons, has high exosome activity, has the function of the signal element 7A protein, can achieve the effects of rapidly promoting the regeneration of the nerve axons and inducing the regeneration of the nerve axons through activating the signal path of the integrin/MAPK, and has strong capacity of inducing the regeneration of the nerve axons; the natural spinal cord decellularized matrix component can simulate the tissue microenvironment of the spinal cord, and has good tissue compatibility and extremely low immune rejection reaction; the slow-release exosome maintains the biological activity, can play a therapeutic role brought by stem cell therapy to a great extent, and avoids related adverse reactions brought by stem cell transplantation, such as immune rejection and the like.

Description

Containing CD56 + Biological material of exosome from subcellular group and preparation method thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a material containing CD56 ⁺ A biological material of exosomes derived from subcellular groups and a preparation method thereof.

Background

Spinal cord injury (Spinal Cord Injury, SCI) is a serious traumatic disorder of the central nervous system. After spinal cord injury, nerve function is lost and the patient is seriously disabled. At present, the treatment such as spine fixation, intramedullary decompression, hormone application and the like is mainly adopted clinically to reduce secondary injury; many therapeutic approaches such as cell therapy, nerve factor and gene therapy are in experimental stages.

Several studies have shown that mesenchymal stem cell transplantation can promote functional recovery from spinal cord injury under conditions of aging, brain injury, neurodegenerative disease, and the like. The main sources of the mesenchymal stem cells in clinic at present are bone marrow, umbilical cord and the like. Compared with bone marrow-derived mesenchymal stem cells, human umbilical cord tissue is used as medical waste, and the Huatong glue content is rich. However, stem cell therapy has many problems to be solved in conventional clinical treatments, including low survival rate of transplanted cells, dedifferentiation of cells, formation of tumors, and the like. There is a great need to find a therapeutic means and method that can exert positive therapeutic effects on stem cells while also reducing the associated risks.

Exosomes (Exosomes) are nanoscale vesicles secreted by a variety of cells, which carry in vivo a variety of bioactive components such as maternal cellular nucleic acids and mirnas, proteins, and the like. The exosome implantation can also avoid risks such as tumors caused by direct stem cell transplantation and a plurality of medical ethical problems related to autologous neural stem cell transplantation. The stem cell-derived exosome has similar functions as stem cells, can effectively promote tissue repair and regeneration, has strong protective capability in the field of central nervous system injury repair, and opens a new way of cell-free treatment.

At present, embryonic Stem Cells (ESCs) and induced pluripotent stem cells (iPS) with full differentiation potential can regenerate nerves, but ESCs are involved in ethics and teratoma, and iPS is involved in the risks of tumorigenesis, immune rejection and the like, and cannot be used clinically.

There is an urgent need for a material that promotes regeneration of nerve axons to repair and regenerate nerves at spinal cord injury.

Disclosure of Invention

The present application provides a CD 56-containing composition ⁺ A biological material of exosomes derived from subcellular groups and a preparation method thereof, which aim to solve the technical problem of how to promote regeneration of nerve axons at spinal cords.

In a first aspect, the present application provides a CD 56-containing composition ⁺ A biological material of a subcellular population-derived exosome comprising: methacrylic acid anhydride gelatin, spinal cord acellular matrix and exosomes; the exosomes are derived from a polypeptide bearing CD56 ⁺ A labeled cell subpopulation; the exosome expresses the signalin 7A protein.

Methacrylic acid acylated gelatin (GelMA) is a photosensitive biological material, and can be rapidly crosslinked and solidified under blue light or ultraviolet light when being matched with a photoinitiator to form a three-dimensional structure with certain strength, and the structure is provided with a cell adhesion site and a matrix metalloproteinase hydrolysis site, so that proliferation and migration of cells can be well supported, and a plurality of cells such as tumor cells, myocardial cells, chondrocytes and the like can be loaded. The mechanical property of the crosslinked hydrogel can be adjusted by changing the concentration of the GelMA material, and the exosome activity can be maintained and improved when the exosome is loaded, and meanwhile, the slow release effect of the exosome is ensured.

The spinal cord decellularized matrix is extracellular matrix obtained by artificial extraction and cell removal of spinal cord tissue, and has the advantage of natural biological material. The spinal cord decellularized matrix has various components, and the IV type collagen, laminin and proteoglycan can be adhered with a large amount of important factors for promoting tissue repair of nerve cells, blood vessels and the like, create a microenvironment suitable for cell growth of nerve axons and the like, promote metabolism and proliferation of cells, moderately degrade and absorb, promote regeneration of the nerve axons and reconstruct the continuity of spinal cord.

The exosome is CD56 ⁺ Human umbilical cord hua tong gum mesenchymal stem cell (Human umbilical cord Wharton's jelly mesenchymal stem cells, HUCWJMSC) exosomes; derived from a CD56 bearing ⁺ The marked cell subset, the surface of which expresses CD56 protein, especially compared with exosomes derived from other cells, highly expresses signalin 7A, and can enhance the regeneration and repair effect on nerve axons by activating integrin/MAPK signal channels.

Preferably, the biomaterial of an embodiment of the present application comprises: in mass fraction, 10% GelMA, 5% acellular matrix, 0.4% photoinitiator, and exosomes with a final concentration of 100 μg/mL.

In the embodiment of the application, the methacrylic acid anhydride gelatin, the spinal cord acellular matrix and the subcellular exosomes are combined to form a biological material, the methacrylic acid anhydride gelatin is taken as a carrier of the spinal cord acellular matrix and the subcellular exosomes to form a slow-release system, the spinal cord acellular matrix is taken as a natural component to provide microenvironment for the adhesion of endogenous nerve cells on one hand, and on the other hand, the spinal cord acellular matrix and the subcellular exosomes are cooperated to exert the effect of inducing the regeneration of nerve axons.

In some embodiments, the biomaterial further comprises a photoinitiator, in particular lithium phenyl-2, 4, 6-trimethylbenzoyl phosphite, in an amount of 0.25-0.5% by mass of the biomaterial.

In the embodiment of the application, the mass fraction of the photoinitiator in the biological material is controlled to be 0.25-0.5%, so that the method has the positive effect of rapid photocuring and can avoid adverse effects such as chemical toxicity and the like.

In the examples of the present application, CD56 is contained ⁺ The biological material of the exosome of subcellular group source can be used as biological ink to prepare biological scaffold.

In an embodiment of the first aspect of the present application, the mass concentration of the exosomes in the biomaterial is 100-200ug/mL, preferably 100ug/mL.

In an embodiment of the first aspect of the present application, the mass fraction of methacrylic anhydride gelatin in the biomaterial is 10% -15%.

In the embodiment of the application, the mass fraction of the methacrylic anhydride gelatin in the biological material is controlled to be 10% -15%, the biological material has the positive effect of rapid gel formation, and the effect that the mechanical hardness is too high and the regeneration of nerve axons is not facilitated can be avoided.

In an embodiment of the first aspect of the present application, the mass fraction of acellular matrix in the biological material is between 5 and 10%.

In the embodiment of the application, the mass fraction of the acellular matrix in the biological material is controlled to be 5-10%, so that the biological material has the positive effect of effectively inducing regeneration of nerve axons, and adverse effects such as gel formation failure can be avoided.

In an embodiment of the first aspect of the present application, the CD56 ⁺ The labeled cell subset is derived from human umbilical cord Wharton's jelly mesenchymal stem cells.

In an embodiment of the first aspect of the present application, the biomaterial has a photo-setting property.

In an embodiment of the first aspect of the present application, the diameter of the exosomes is +.150 nm.

In an embodiment of the first aspect of the present application, the biomaterial is applied to the damaged spinal cord site in the form of a dressing, and the biomaterial is used in an amount of 80-100ul/cm ² 。

In an embodiment of the first aspect of the present application, the exosomes are present in an amount of 1.5X10 in the local area of the injured spinal cord ⁷ ～2.5×10 ⁷ Individual/cm ² 。

In the embodiment of the application, the number of the exosomes in the local part of the injured spinal cord is controlled to be 1.5X10 ⁷ ～2.5×10 ⁷ Individual/cm ² The active effect of promoting the regeneration of the axon can be exerted, and adverse reactions can be avoided.

In an embodiment of the first aspect of the present application, the spinal cord decellularized matrix composition comprises: type IV collagen, laminin and proteoglycans.

In the examples of the present application, the components of spinal cord decellularized matrix include type IV collagen, laminin and proteoglycan, and the synergistic effect of the different components can induce regeneration of nerve axons.

In a second aspect, the present application provides a CD 56-containing composition ⁺ A method for preparing a biological material of a subcellular population-derived exosome, the method comprising the steps of:

obtaining a human umbilical cord sample after parent separation;

sorting cells in the human umbilical cord Huatong gel sample to obtain a marker CD56 ⁺ Mesenchymal stem cell subpopulations of (a);

culturing the mesenchymal stem cell subgroup and harvesting cell supernatant;

filtering, separating and re-suspending the cell supernatant to obtain exosomes;

detecting the protein content in the exosomes to obtain exosomes containing target protein content;

obtaining spinal cord decellularized matrix;

mixing the exosomes, spinal cord acellular matrix and methacryloyl gelatin, and incubating to obtain the biological material.

Specifically, the method comprises the steps of: (1) Weighing a proper amount of GelMA and a photoinitiator, adding the GelMA and the photoinitiator into phosphate buffer saline (Phosphate buffer saline, PBS) and dissolving the GelMA and the photoinitiator at 60 ℃ for 40min;

(2) Weighing a proper amount of spinal cord acellular matrix, adding the acellular matrix, stirring and dissolving at 37 ℃,

(3) CD56 addition to the spinal cord decellularized matrix bio-ink ⁺ HUCWJMSC exosomes, so that the final concentration reaches the expected concentration to obtain the target biological material;

in some embodiments, a method of preparing a biomaterial may include:

(1) 1g of methacrylic anhydride gelatin and 0.04g of photoinitiator are weighed and added into 10mL of PBS, and stirred and dissolved for 40min at 60 ℃;

(2) Adding 0.05g spinal cord acellular matrix into the obtained solution, stirring at 37 ℃ for dissolution;

(3) Addition of CD56 to the spinal cord decellularized matrix bioink ⁺ HUCWJMSC exosomes such that their final concentration is 100 μg/mL;

in an embodiment of the second aspect of the present application, the target protein is signalin 7A.

CD56 ⁺ Acquisition of HUCWJMSC

Specifically, the method comprises the steps of: (1) Fresh umbilical cord specimens are taken, PBS is fully washed until no blood stain exists, the umbilical cord specimens are cut into segments with the length of 3-4 cm, and the amniotic membrane, umbilical vein and umbilical artery are separated to obtain the Huatong gel. Cutting Huatong gel into 1mm ³ The fragments are added into 1g/L type II collagenase, and after continuous digestion for 30min by a constant temperature shaker at 37 ℃, continuous digestion for 30min by a constant temperature shaker at 37 ℃ of 0.25% pancreatin is carried out immediately. The digestions were filtered through a 70um cell sieve, the filtrate was centrifuged at 400G for 5min, and the supernatant was discarded and washed 2 times with PBS.

(2) Cells were resuspended using 200 μl FACS buffer (2% pbs) according to 2:100, adding a blocking solution, incubating for 10min, centrifuging at 400G for 5min, and collecting precipitate.

(3) The pellet was resuspended in 100. Mu.L FACS buffer, 0.1ul zombie viable dead dye was added, incubated at room temperature for 15min, and centrifuged at 400G for 5min before harvesting the pellet.

(4) The pellet was resuspended using 100 μ L standing buffer according to 1:100 were added to prepare CD31, CD235a, CD45, CD90, CD73 and CD56 antibodies and incubated for 30min in the absence of light.

(5) The cells were resuspended by centrifugation at 400G for 5min using 500. Mu.L FACS buffer.

(6) Filtering the cell suspension into single cell suspension by using a cell sieve, and performing on-machine detection to obtain CD56 ⁺ HUCWJMSC。CD56 ⁺ Extraction of HUCWJMSC exosomes

(1) When CD56 ⁺ When HUCWJMSC grows to about 80% density, the culture supernatant is discarded, and after washing with PBS for 2 times, the culture is continued for 48 hours by changing MEM-alpha medium containing 10% of exosome-free FBS, and the cell supernatant, that is, the conditioned medium, is collected. The conditioned medium is then treated as follows:

(2) The conditioned medium was centrifuged at 500G for 10min at 4℃and the supernatant was collected.

(3) The supernatant was collected by centrifugation at 2000G at 4℃for 30min.

(4) Centrifuging at 4deg.C and 10000G for 60min, and collecting supernatant.

(5) The supernatant was filtered using a 0.22 μm pore size filter.

(6) Centrifugation at 110000G for 90min at 4℃and careful aspiration of supernatant, pre-chilled PBS resuspension pellet.

(7) Centrifugation at 110000G for 90min at 4deg.C, careful aspiration of PBS, addition of 100-200 μL of pre-chilled PBS, resuspension of bottom pellet, sub-packaging, and storage at-80deg.C.

In an embodiment of the second aspect of the present application, the sorting sorts the mesenchymal stem cell subpopulations by flow cytometry.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the biomaterial provided by the embodiment of the application comprises: methacrylic acid anhydride gelatin, spinal cord acellular matrix and exosomes; the exosomes are derived from a subpopulation of cells bearing a cd56+ tag; the exosome expresses signalin 7A protein; among exosomes extracted from the extract, exosomes express a plurality of protein types for promoting regeneration of nerve axons, have high exosome activity, and have signalin 7A protein, and can achieve the effects of rapidly promoting regeneration of nerve axons and inducing regeneration of nerve axons through activating an integrin/MAPK signal path, and have strong capacity; the natural spinal cord decellularized matrix component can simulate the tissue microenvironment of the spinal cord, and has good tissue compatibility and extremely low immune rejection reaction; the slow release system is formed after light solidification, the slow release exosome keeps the biological activity, the therapeutic effect brought by stem cell therapy can be exerted to a great extent, and meanwhile, the related adverse reactions brought by stem cell transplantation, such as immune rejection and the like, are avoided; when in use, the photoinitiator is added for curing. .

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 shows a human umbilical cord Wharton's jelly mesenchymal stem cell subset (CD 56) ⁺ HUCWJMSCs);

fig. 2 is an extraction and identification diagram of human umbilical cord hua tong gum mesenchymal stem cell subgroup exosomes provided in the examples of the present application;

FIG. 3 is a schematic diagram of a CD56 provided in an embodiment of the present application ⁺ HUCWJMSC-EXOs promote the function recovery of spinal cord injured mice;

FIG. 4 is a schematic diagram of a CD56 provided in an embodiment of the present application ⁺ Influence of HUCWJMSC-EXOs on regeneration of nerve axons;

FIG. 5 is a diagram of a decellularized characterization of spinal cord decellularized matrix prepared according to a method of one embodiment of the invention;

FIG. 6 shows a slow release profile of a biomaterial to an exosome according to one embodiment of the invention;

fig. 7 is a functional recovery chart of the biological material provided in the embodiment of the present application on a spinal cord injury mouse.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present application based on the embodiments herein.

Umbilical cord samples used in the examples of this application were obtained from healthy mothers after delivery, and the study was approved by the ethical review Committee of the Xiangya Hospital at the university of south China and informed consent was obtained from all participants prior to sample collection; laboratory instruments and reagents are commercially available.

Example 1

Human umbilical cord hua tong gum mesenchymal stem cell CD56 ⁺ Subpopulation (CD 56) ⁺ HUCWJMSC).

The study was approved by the ethical review board of the Xiangya Hospital, university, south China, and informed consent was obtained from all participants prior to sample collection. Human umbilical cord samples were obtained from healthy mothers after delivery and processed within 6 hours after collection. The umbilical cord was first washed twice with Phosphate Buffered Saline (PBS) containing 100U/mL penicillin and 100. Mu.g/mL streptomycin to remove the expected residual blood. The washed umbilical cord was cut into 3-4 cm long pieces in a petri dish, and the amniotic membrane was separated from the umbilical vein and umbilical artery to obtain Huatong gel. Then cutting Huatong glue into 1mm ³ The fragments are added into 1g/L type II collagenase, and after continuous digestion for 30min by a constant temperature shaker at 37 ℃, continuous digestion for 30min by a constant temperature shaker at 37 ℃ of 0.25% pancreatin is carried out immediately. The digestions were filtered through a 70um cell sieve, the filtrate was centrifuged at 400G for 5min, and the supernatant was discarded and washed 2 times with PBS.

Separation of CD56 by flow cytometry ⁺ HUCWJMSC, firstly separating some impurity cells by using general technique, then using CD73 antibody and CD90 antibody to implement flow cytometry to separate human umbilical cord HuatongAnd (3) glue mesenchymal stem cells. CD56 ⁺ HUCWJMSC represents a cell subset highly enriched for CD56 human umbilical cord Wharton's jelly mesenchymal stem cells, wherein HUCWJMSC represents human umbilical cord Wharton's jelly mesenchymal stem cells.

The anti-CD 56 antibody is utilized according to the requirement, and a flow cytometry method is implemented to separate the mesenchymal stem cell subgroup of the human umbilical cord Wharton's jelly. Human umbilical cord hua tong gum mesenchymal stem cell subgroup marker is CD56 ⁺ 。

Human umbilical cord Wharton's jelly mesenchymal stem cell subpopulations were transferred to DMEM/F-12 and supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100 μg/mL streptomycin.

As shown in fig. 1, 6 diagrams in fig. 1 are given arrow marks in order: (1) removing debris to obtain a cell process map (abscissa is the relative intensity of the area signal under the forward scattered light-curve, reflecting the volume of the cell, ordinate is the relative intensity of the side scattered light signal, reflecting the granularity of the cell), (2) obtaining a single cell map (in-frame obtaining a higher ratio of single cells, abscissa is the relative intensity of the area signal under the forward scattered light-curve, ordinate is the relative intensity of the forward scattered light-width signal), (3) obtaining a living cell map (in-frame obtaining living cells, out-of-frame obtaining dead cells; DAPI is 4', 6-diamidino-2-phenylindole, abscissa is the relative intensity of the DAPI fluorescent signal, ordinate is the relative intensity of the side scattered light signal, reflecting the granularity of the cell), (4) obtaining CD45/235a/31 ^- Cell map (the relative intensity of fluorescence signal of the horizontal axis is CD45/235a/31, the vertical axis is the relative intensity of side scattering light signal, reflecting the granularity of cells, endothelial cells, immune cells and blood cells are removed in the process), (5) HUCWJMSC cell map (the human umbilical cord Wharton's jelly mesenchymal stem cells in the frame, the horizontal axis is CD 90) ⁺ Relative intensity of fluorescent signal, ordinate CD73 ⁺ Relative intensity of fluorescent signal, CD90 ⁺ And CD73 ⁺ Is a marker of HUCWJMSC), (6) a map of CD56+HUCWJMSC (in-frame with CD 56) ⁺ Labeled cell subpopulations with CD56 on the abscissa ⁺ Fluorescent signal relative intensity, and ordinate is side scatter light signal relative intensity), wherein the portion circled by the figures is the target cell, and 6 figures are the process of obtaining target subcellular groupThe method comprises the steps of carrying out a first treatment on the surface of the The positive rate of markers CD73 and CD90 of stem cells of the sorted cells is 25.5 percent (the positive rate can be directly measured from the graph); the CD56 positive rate of the sorted cells is 6.57%, and the CD56 is obtained by two positive rates ⁺ A HUCWJMSC subset.

In the prior art, only human umbilical cord Wharton's jelly mesenchymal stem cells are usually obtained, but the human umbilical cord Wharton's jelly mesenchymal stem cell subgroup with specific markers is not obtained, but the human umbilical cord Wharton's jelly mesenchymal stem cells with CD56 in the application ⁺ The marked cell subset, the exosomes extracted from the marked cell subset, has significantly stronger capacity of promoting regeneration of nerve axons compared with other unsorted human umbilical cord Wharton's jelly mesenchymal stem cells through research.

Example 2

CD56 in human umbilical cord huatong gel mesenchymal stem cells ⁺ Preparation of cell subpopulations exocrine

Waiting for CD56 ⁺ When the cell subset grows to 60 percent 70 percent confluency, collecting culture supernatant, filtering by a 0.22 mu m filter membrane, and centrifuging at the temperature of 4 ℃ and the temperature of 500G for 10 minutes; centrifuging at 4 ℃ for 30min at 2000 g; centrifuging at 4deg.C and 10000g for 60min; after centrifugation at 110000g for 90min, the supernatant was discarded and the pellet was resuspended using phosphate buffer; centrifuging again at 110000g for 90min, discarding supernatant, re-suspending with small amount of phosphate buffer solution, precipitating to obtain exosomes, and storing at-80deg.C, in figure 2, under A. Microscope CD56 ⁺ The labeled cell subpopulation was cultured for 7 days with a microscope magnification of 100×, and CD56 after sorting was illustrated by the figure ⁺ The labeled cell subpopulation has the characteristic of a long spindle shape of a typical mesenchymal stem cell. B. CD56 for transmission electron microscope observation ⁺ HUCWJMSC-EXOs morphology. C. CD56 acquisition using Nanoparticle Tracking Analysis (NTA) ⁺ Particle size distribution and images of HUCWJMSC-EXOs, the abscissa represents the particle size of exosomes, and the ordinate represents the relative concentration. D Western Blot (WB) analysis of exosomes specific markers showed a clear presence of CD63 and TSG 101.

Example 3

Ability of subgroup exosomes to promote functional repair of mouse spinal cord injury model

All animal handling was authorized by the university of south-middle animal ethics committee. Conventional anesthesia, skin preparation, disinfection and drape. The skin, subcutaneous, muscle and lamina are sequentially cut with the T10 spinous process as the center, revealing the spinal cord. The dorsal half-cut model was created by dorsal cutting 1 mm. The wound is irrigated, the incision is closed layer by layer, and the dressing covers the wound and is secured. All mice are fed into an animal laboratory, the ambient temperature is 22-24 ℃, and the relative humidity is 60-80%. After operation, the feed is fed in separate cages, and the feed and the drinking water can be fed by themselves. Penicillin 2WU/Bid was routinely injected intramuscularly 3d post-operatively to combat infection. The bladder is squeezed 2-4 times per day to help urinate until the urination reflex is recovered.

The motor function and motor coordination and stabilization functions of the hind limbs of the mice after spinal cord injury are evaluated by using a BMS (Basso Mouse Scale) main scoring system and a secondary scoring system respectively at 1,3,7, 14, 21, 28,42 and 56 days after spinal cord injury, wherein the BMS main scoring is 9 points altogether, from 0 point to 9 points, and the motor function recovery is better when the score is higher; the BMS sub-scores a total of 11 points, the higher the score the better the exercise stability and coordination. Each mouse was double-blinded for 5 minutes in the experimental design by two researchers familiar with BMS scoring. The average score for each mouse was then recorded.

The abscissa of panel a in fig. 3 is Pre-sur (time point before spinal cord injury), SCI 1D, SCI 3D, SCI 7D, SCI 14D, SCI 21D, SCI28D, SCI D and 56D ( time points 1,3,7, 14, 21, 28,42 and 56 days after spinal cord injury in sequence), and the ordinate represents BMS major score, from 0 to 9 points, the higher the score, the better the recovery of hindlimb motor function of the mice is illustrated; panel B shows the abscissa of Pre-sur (time point before spinal cord injury), SCI 1D, SCI 3D, SCI 7D, SCI 14D, SCI D, SCI 3828D, SCI42D and 56D ( time points 1,3,7, 14, 21, 28,42 and 56 days after spinal cord injury in sequence), and the ordinate represents the BMS major and minor score, from 0 to 11 points, the higher the score, the better the hind limb exercise stability and coordination recovery of the mice are illustrated; 0/50/100/150/200 in the figure represents the different concentrations of exosomes (in ug/mL), respectively.

As can be seen from FIG. 3, CD56 is 100-200. Mu.g/ml ⁺ HUCWJMSC-EXOs treatment can significantly improve the motor function of the double lower limbs and the stability and coordination of the motor after the spinal cord injury of the mice.

Example 4

Exosomes promote the regeneration ability of nerve axons

Pregnant mice were dissected for 13-14 days, the gestation sacs were removed, and after digestion, centrifugation and resuspension of fetal mouse brain cortex tissue, complete medium (high sugar DMEM medium, 10% fbs, 1% penicillin/streptomycin double antibody) was used and inoculated into 6-well plates pre-coated with polylysine. After 4 hours the neuron-specific medium (Neuroblast medium containing 2% B27,0.24% GLUTA-MAX and 100U/mL penicillin and 100. Mu.g/mL streptomycin) was changed and the medium was changed once every three days. Cortical neuronal cells were treated with the subpopulations of exosomes (concentrations 100-200 μg/ml) prepared in example 2, unsorted HUCWJMSC-EXOs (concentrations 100-200 μg/ml) and PBS (control group), fixed after 3 days, stained with Tuj-1, and observed for axon regeneration. As can be seen from fig. 4, the subgroup exosomes of the present application have a greater ability to promote regeneration of nerve axons.

Example 5

Preparation of spinal cord acellular matrix

(1) Taking a 12-week-old SD female rat, removing neck, killing, cutting back skin, removing muscle and ligament, exposing vertebral canal, removing vertebra with vertebral plate forceps, completely taking out thoracic spinal cord tissue, placing into a culture dish containing sterile normal saline, and carefully stripping hard film and surface blood vessel with micro forceps and micro scissors to obtain fresh thoracic spinal cord tissue.

(2) Washing the obtained spinal cord tissue with PBS for 3 times, 30min each time, and wrapping with gauze; freezing spinal cord tissue in liquid nitrogen for 2min, thawing in a constant-temperature water bath kettle at 37 ℃ for 10min, and circulating for 5 times; subsequently rinsed with 1% diabody/PBS for 30min, repeated 3 times, digested in nuclease solution at 37℃for 2 hours; the sample is ground into powder by a freeze-drying and ball-milling instrument, and is preserved in a refrigerator at the temperature of minus 80 ℃ after being disinfected.

(3) DAPI staining of decellularized spinal cord tissue sections: spinal cord decellularized tissue is taken, 4% paraformaldehyde is fixed for 12 hours, and gradient dehydration is carried out by 20% and 30% sucrose sequentially. OCT was embedded and sectioned in a frozen microtome at a thickness of 16. Mu.m. After rewarming the sections for 1h, washing the sections with PBS for 3 times and 3min each time, dripping DAPI staining solution at the specimens, incubating the sections for 3min at room temperature in a dark place, and taking images by using a fluorescence microscope after sealing the sections. As can be seen from FIG. 5, the spinal cord acellular matrix has low DNA content and good acellular effect.

Example 6

Construction and application of biological ink containing subgroup exosomes

(1) Preparation of acellular matrix bio-ink: 1g of methacrylic anhydride gelatin and 0.04g of photoinitiator are weighed and added into 10mL of PBS, and stirred and dissolved for 40min at 60 ℃; to the above obtained solution, 0.02g of spinal cord acellular matrix was added, and the mixture was dissolved by stirring at 37 ℃.

(2) Preparation of biological ink containing subgroup stem cell exosomes: cd56+ HUCWJMSC exosomes were added to the spinal cord decellularized matrix bio-ink to a final concentration of 100 μg/mL.

(3) Constructing a mouse spinal cord dorsal half-cutting model, heating the obtained biological ink containing the subgroup stem cell exosomes to 37-40 ℃ to make the biological ink be in a water phase, and locally coating the damaged spinal cord according to 100ul/cm ² The subgroup exosome-gel slow release preparation was covered, and in addition, PBS-gel preparation, HUCWJMSC-EXOs-gel preparation and spinal cord decellularized matrix-gel preparation were covered as controls, respectively. At 365 405nm60mW/cm ² The hydrogel is changed from water phase to solid phase by illumination for at least 30s, and the wound is sutured. CD56 ⁺ HUCWJMSC-EXOs represent CD56 ⁺ Human umbilical cord hua tong gum mesenchymal stem cell exosomes.

(4) Determination of exosome release: the photocured biological ink containing the stem cell exosomes of the subpopulation is placed in a Transwell upper chamber (8 μm), 150 μl of PBS is added in a lower chamber, and the mixture is placed in an incubator at 37 ℃ for incubation. At a predetermined time, 15. Mu.L of PBS was removed from the lower chamber and an equal volume of PBS was added. Protein content of PBS removed at each time point was measured using micro BCA kit, and the release percentage of exosomes was calculated.

In fig. 6, the abscissa indicates time, and the ordinate indicates the exosome release rate, and the higher the value, the more exosome release.

As can be seen from FIG. 6, the biomass containing the stem cell exosomes of the subpopulation can stably release exosomes for more than 24 days.

(5) The therapeutic effect of subpopulation-containing stem cell exosome biomaterials on motor function of hind limbs in mice following spinal cord injury was assessed on the basis of example 3 using a BMS scoring system at days 1,3,7, 14, 21, 28,42 and 56 following spinal cord injury.

As can be seen from FIG. 7, the meanings of the abscissa and the ordinate are the same as those of FIG. 3, and in the graph, A. The biological material containing the stem cell exosomes of the subpopulation can remarkably improve the movement function of the double lower limbs after the spinal cord injury of the mice. B. The exosome biological material containing the sub-population stem cells can obviously improve the stability and coordination of the movement of the double lower limbs after the spinal cord injury of the mice.

Furthermore, to investigate the effect of different variable concentrations on bio-ink, the inventors performed several sets of control experiments on the basis of this example, the specific variations of the experimental variables being shown in the following table:

variable name	First group of	Second group of	Third group of	Unit (B)
					GelMA	10％	12.5％	15％	Mass percent
DCM	5％	7.5％	10％	Mass percent
					Photoinitiator	0.25％	0.4％	0.5％	Mass percent
Final exosome concentration	100	150	200	μg/mL

Each variable was chosen as a specific number, for example:

GelMA (12.5%), DCM (5%), photoinitiator (0.4%), and CD56+HUCWJMSC exosomes with a final concentration of 100 μg/mL were used as one experimental group, and experimental results showed that biological ink could be obtained in these experimental protocols, and biological materials could be used to obtain the results for regeneration of nerve axons, and the performance test results were similar to the previous examples, and the experimental results were repeated more and not repeated.

Experiments prove that when the mass concentration of the exosomes is 100-200ug/mL, compared with other concentrations, the exosomes have better effect of promoting the recovery of the nerve axon function. The CD56 was confirmed by BMS scoring and Tuj-1 staining experiments ⁺ The subgroup exosomes have better effects of promoting spinal nerve function recovery compared with unsorted cell exosomes, due to the stronger ability of promoting axon regeneration.

As proved by BMS scoring system experiments, the biological material is used for coating the local part of the injured spinal cord in a dressing form, and the dosage of the exosome hydrogel mixed system is 80-100ul/cm ² . At this time in the local area of the injured spinal cordThe number of active exosomes is approximately 1.5x10 ⁷ ～2.5×10 ⁷ Individual/cm ² The effect of promoting the recovery of spinal nerves can be achieved by promoting the regeneration of nerve axons to about 70% before injury.

It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. CD 56-containing material _⁺ A biological material derived from exosomes derived from a subcellular population, comprising: methacrylic acid anhydride gelatin, spinal cord acellular matrix and exosomes; the exosomes are derived from a polypeptide bearing CD56 _⁺ A labeled cell subpopulation; the exosome expresses signalin 7A protein; the diameter of the exosome is less than or equal to 150nm; the exosomes are concentrated in mass in the biological materialThe degree is 100-200ug/mL.

2. The biomaterial of claim 1, wherein the mass fraction of methacrylic anhydride gelatin in the biomaterial is 10% -15%; and/or the number of the groups of groups,

the mass fraction of the acellular matrix in the biological material is 5-10%.

3. The biomaterial of claim 1, wherein the CD56 _⁺ The marked cell subset is derived from human umbilical cord Wharton's jelly mesenchymal stem cells;

and/or the biomaterial has a photosensitive curing property.

4. The biomaterial according to claim 1, wherein the biomaterial is applied to the damaged spinal cord part in the form of a dressing, and the biomaterial is used in an amount of 80-100ul/cm _² 。

5. The biomaterial of claim 1, wherein the exosomes are present in an amount of 1.5 x 10 in the injured spinal cord part ⁷ ～2.5×10 ⁷ Individual/cm _² 。

6. The biomaterial of claim 1, wherein the spinal cord decellularized matrix component comprises: type IV collagen, laminin and proteoglycans.

7. A CD 56-containing film as claimed in any one of claims 1 to 6 _⁺ A method for preparing a biological material derived from exosomes derived from a population of subcellular cells, the method comprising the steps of:

obtaining a human umbilical cord sample after parent separation;

sorting cells in the human umbilical cord sample to obtain a marker CD56 _⁺ Mesenchymal stem cell subpopulations of (a);

culturing the mesenchymal stem cell subgroup and harvesting cell supernatant;

filtering, separating and re-suspending the cell supernatant to obtain exosomes;

detecting the protein content in the exosomes to obtain exosomes containing target protein content;

obtaining spinal cord decellularized matrix;

mixing the exosomes, spinal cord acellular matrix and methacryloyl gelatin, and incubating to obtain the biological material.

8. The method of claim 7, wherein the protein of interest is signalin 7A.

9. The method of claim 7, wherein the sorting sorts the mesenchymal stem cell subpopulations by flow cytometry.

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